Convertases Introductionstructure Of Integrins

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Integrins are heterodimeric adhesion molecules that link the extracellular matrix to the actin cytoskeleton and signaling cascades. Integrins receive signals from other receptors, leading to the activation of ligand binding (inside-out signaling) and to matrix assembly. Upon binding ligands, integrins also activate intracellular signaling pathways (outside-in signaling). In this way, cell adhesion is coordinated with other events to orchestrate complex cell behaviors, such as cell migration and/or invasion.

A-Majid Khatib (ed.), Regulation of Carcinogenesis, Angiogenesis and Metastasis by the Proprotein Convertases, 107-119. © 2006 Springer.

In mammals, the integrin receptor family includes at least 18 different a subunits and 8 P subunits that can associate to form 24 distinct integrins. As both a and P subunits recognize the ligand, each aP association presents its own ligand specificity. In a structural feature, both subunits present a large extracellular domain, a transmembrane segment and a short intracellular region; except for P4 the only subunit bearing a large cytoplasmic domain.

Since 1987 when Hynes spoke about integrin for the very first time [1], a lot of structural information has been collected (for recent reviews, see [2-4]. Electron microscopy for a5p1 and aIIbp3 [5-8], nuclear magnetic resonance [9] or the mapping of the epitopes structures of conformation sensitive and activating antibodies lead to a global structural model. However, the major advance in our understanding of the relation between structure and function of integrins was provided by the X-ray crystal structure of the extracellular regions of avP3 integrin [10, 11].

Integrin a subunits are separated into 2 categories: a subunits that undergo an endoproteolytic processing, like avP3, and a subunits bearing an inserted (I) domain, like a1P1 (Figure 1). The avP3 integrin presents 12 domains that form an ovoid head and two tails or legs. In the crystal, avP3 is severely bent, reflecting an important flexibility that may be linked to integrin regulation [10]. In the bent state,

Structure Integrins
Figure 1. Comparative structure of integrins with cleaved (left) or uncleaved (right) subunit. Modified from refs [2, 3]

the ligand-binding domains in the headpiece would be closely juxtaposed to the membrane-proximal portions of the stalks. There is now increasing evidences that conformational changes are propagated all along the molecule and that interactions between the a and P cytoplasmic tails are critical for stabilizing the association between a and P extracellular regions. Disruption of these intracellular interactions by inside-out signals leads to extension of the integrin, repositioning the head region to point away from the cell surface [7].

Half of the 18 integrin a subunits contain an additional I domain inserted into the P-propeller. Where present, this domain is the major site of ligand binding. It is now known how the I domain interacts with ligands. The interaction is due to cooperation between the I domain on a subunit (aI) and an I-like domain located on P subunit (PI domain). The PI domain contains a metal ion-dependent adhesion site (MIDAS) positioned to participate in a ligand interface with aI domain. The aI domain can adopt two stable conformations (closed and open) leading, respectively, to low or high affinity state of the integrin [12, 13].

1.1 Consensus Site for Endoproteolytic Processing of Integrin a Subunit

Among the different integrin a subunits identified to date, a3, a4, a5, a6, a7, a8, a9 aE, av and aIIb subunits are proteolytically cleaved during their biosynthesis. For the majority of a subunits, the maturation occurs near the carboxyl-terminal part of the extracellular domain, resulting in a heavy chain (about 125 kDa) that is disulphide-linked to a membrane spanning light chain (about 25 kDa). In contrast, the a4 subunit can be expressed at the cell surface either intact or cleaved near the middle of the molecule into non disulphide-linked fragments of 80 and 70 kDa. In the case of aE, the cleavage take place near the NH2-terminal instead of the COOH-terminal as for other a subunits [14]. The sequence of cleavage regions suggests the existence of two groups that present distinct consensus cleavage sites: Arg-X-(Lys/Arg)-Arg I for a3, a6, a7, a9, aE (group I) and His-X-X-X-(Lys/Arg)-Arg I for a4, a5, a8, av, and aIIb (group II) (Figure 2).

The presence of an arginine at P4 position or a histidine at P6 position is thus a distinguishing feature of each group. The aIIb subunit has been classed in the group II, although it exhibits both Arg at P4 and His at P6 [15].

1.2 Cleavage Across the Evolution

Integrins are present across the evolution from the nematode Caenorhabditis. elegans to mammals. At least three Drosophila melanogaster integrins a subunits (aPS1, aPS2 and aPS3) present an endoproteolytic cleavage leading to the formation of a light and a heavy chain linked by a disulphide bond. In addition, C. elegans a1 and a2 subunits are also cleaved [16].

Phylogenetic studies of integrin a subunits have shown that I domain-containing subunits form a monophyletic group apart from cleavable a subunits [16, 17].

Group I

Group II


Gly Pro a 5 Ser Lai ag lie Pro

V av Arg Asp


































Glii Arg Ai u^ AJa Leu

Lys Lys Ara^Glu lie

Arg Ai"g Arg Glu Leii

Lys Arg Ara^Val Gin

Asp Arg Arg^Gln He

Ser Lys Arg1 Ser Tlir

Gill Lys Arg^ Glu Ala

Arg Lys Arg

Glii Val

Thr Lys Al"g Asp Leu

Figure 2. Alignment of consensus cleavage sites in integrin a subunits from mammals

The I domain family forms two main clusters that contains no known invertebrate members. Cleavable a subunits are gathered into four major clusters. The PS1 cluster contains Drosophila aPS1, a C. elegans sequence and vertebrate a3, a6 and a7. The PS2 cluster includes Drosophila aPS2, a C. elegans sequence, two echinoderm sequences and vertebrate a5, a8, av and aIIb. Interestingly, vertebrate a subunits of the PS1 and PS2 clusters exhibit, respectively, Arg at P4 position or His at P6 in the cleavage region. The PS3 cluster only contains Drosophila sequences, aPS1 and two additional a subunits (aPS4 and aPS5) revealed by complete sequencing of the genome. The a4 and a9 subunits form the fourth cluster of a integrins lacking I domain. The phylogeny thus strongly suggests that the common ancestor of deuterostomes (including vertebrates) and protostomes (including insects) possessed at least 3 types of a subunits corresponding to aPS1, aPS2 and aPS3, the latter being lost in the vertebrate lineage [17]. It is worth noting that invertebrates possess only cleavable a subunits and that only vertebrates display a subunits having the I domain. The cleavage of a subunits was thus maintained during evolution from C. elegans to vertebrates, while the I domain appeared before the divergence of birds and mammals. Because, except for aE, the presence of cleavage and I domain on integrin a subunits are mutually exclusive, the I domain possibly appeared in place of cleavage. Knowing the importance of the I domain in integrin function, these observations strongly suggest that the ancestral cleavage was maintained for functional interest.

1.3 Integrin Cleavage: Which Enzyme? Which Location?

In our laboratory, we studied the location of a6 subunit cleavage. We showed that endoproteolytic cleavage only occurred after integrin heterodimerisation, probably in the trans-Golgi network (TGN) [18]. Some data are in favor of this hypothesis. First, blocking pro-a6 in the ER by brefeldin A treatment completely prevented its endoproteolytic maturation, but not its association with ^4. Second, we have demonstrated that a6 is cleaved after the acquisition of endoglycosidase H resistance (taking place in the cis-Golgi compartment), but before the subunit reached the cell surface [18].

The proprotein convertases (PCs) are a family of endoproteases that activate proproteins by cleavage at basic motifs. Seven PCs are now identified: furin, PC1 (also named PC3), PC2, PACE4, PC4, PC5 (PC6), PC7 (PC8, LPC). The list of substrates activated by these convertases is very vast and includes neuropeptides, peptide hormones, growth and differentiation factors, receptors, adhesion molecules, etc. Observations realised in LoVo cells pointed out the proprotein convertase furin as a possible candidate for integrin cleavage. Indeed, in these cells the gene encoding for furin has a frameshift mutation within one allele and a missense mutation (W547R) within the second allele, resulting in lack of processing activity of the mutant furin [19]. In these furin-deficient cells, integrin subunits a3, a6 and av are not cleaved and this defective pro-a chain processing was rescued by recombinant furin [20].

To answer the question of the cleavage specificity of a subunits by different PCs, Lissitzky and collaborators analyzed the processing of pro-a integrin subunits by restoring furin activity and over-expressing the other convertases in furin-deficient LoVo cells. They conclude that only furin, PC5A and, to a lesser extent, PACE4 are able to carry out endoproteolytic processing of a3, a5, a6 and av subunits [21]. An in vivo study from Stawowy and collaborators confirmed that PC5 is involved in av processing. Indeed, on vascular smooth muscle cells, av and PC5 are co-localised and regulated in the same manner during vascular remodeling [22]. In addition, furin was also found associated with av subunit in renal podocytes [23]. Results obtained for a4^1 integrin are quite the same that for Lissitzky study. Using LoVo cells, authors showed that a4 is processed by PC5A, furin or PACE4, but not by PC7. However, this leukocyte subunit is quite particular, as its cleavage site is positioned at the middle of the extracellular part of the subunit. Moreover, the integrin can be addressed at the cell surface cleaved or uncleaved and the cleavage status can be correlated with lymphocyte activation. It is one of the few example for direct correlation between a subunit cleavage and integrin function [15].

Others proteases could also cleaved av integrin subunit if PCs are deficient. Strongin and collaborators used a synthetic convertase inhibitor in breast carcinoma MCF7 cells co-expressing av^3 integrin and MT1-MMP. They demonstrated that in these conditions, MT1-MMP is capable of processing the precursor of integrin av subunit [24]. This cleavage appears to occur between Cys852 and Cys904 and generates a functional av^3 integrin in term of cell adhesion. MT1-MMP is also able to cleave other PC-cleavable a subunits, such as a3 and a5, but failed in the processing of PC-resistant a2 integrin chain [25].

1.4 Integrin Function in Cancer

Cell adhesion and migration are essential for tumour invasion. The interest of slowing down tumour growth by integrin-dependent stabilization of cell-substrate interactions on fibronectin was recognised in the 1980s. Since these initial reports, integrin up- or down-regulation has been regularly observed in tumour progression (for reviews see [26-30]). Integrins act at different levels during tumour progression:

1) Signalling for the lost of neighbouring cell contacts. In addition to classical cell-cell receptors, such as E-cadherin, several integrins are also involved in this process. Thus, overexpression leads to disruption of adherens junctions in epithelial cells [31]. Reciprocally, p1-blocking antibodies induce reformation of adherens junctions and polarisation of tumour cells [32]. Moreover, functional interactions between E-cadherin and av-containing integrins has been reported in carcinoma cells [33].

2) Migration across the interstitial stroma. Integrins play a key role in the complex process of cell migration. In addition to allowing anchorage necessary for cell displacement, integrins induce signalling ending up to cytoskeleton remodelling. These signalling pathways typically lead to Rho GTPases activation via the phosphorylation of focal adhesion kinase (FAK) [34, 35].

3) Survival in the interior circulation (blood or lymph) by adhesion to platelets [36] or lymphocytes [37].

4) Angiogenesis of distal tumour. av^3 and av^5 integrins have been involved in growth factor- and tumour-induced angiogenesis in multiple animal models (for review [38]). In addition to these largely studied integrins, other members of the family clearly contribute to blood vessel formation, either during development or tumour progression. Thus, mice carrying a targeted deletion of the signalling portion of the integrin ^4 subunit display drastically reduced angiogenesis in response to bFGF in Matrigel plug assay [39]. The a1^1 and a2^2 integrins also regulate angiogenesis induced by VEGF [40, 41].

1.5 Consequences of the Perturbation of Integrin Cleavage on Cancer

The role of endoproteolytic cleavage of integrin a subunits is still unclear, but it may play a role in integrin function. As discussed above, the cleavage is conserved, not only in different a chains but also across species (from invertebrates to mammals), suggesting that it might be of functional importance. Furthermore, post-translational proteolysis is a common mechanism required for the synthesis of many biologically active proteins in bacteria, fungi, yeast, invertebrates and mammals [42].

The cleavage status of integrin a subunits has never been studied in vivo excepted for a6 in differentiating lens fibre cells. Authors reported that expression of the uncleaved form of a6 integrin progressively increased relative to the cleaved form during lens cell differentiation, suggesting that the uncleaved form of a6 integrin may have a unique role in the embryonic lens [43].

We have briefly described above the involvement of integrins in tumour invasion. However, although some cleavable integrins, such as av^3, av^5 or av^6 have a central role in cell migration and angiogenesis, it is impossible to ascertain that cleavable integrins are more important than non-cleavable integrins in these processes and that a subunit cleavage is determinant for tumour progression. To address directly the functional importance of integrin cleavage, different studies have been performed in vitro, either by site-directed mutagenesis of integrin a subunits or by convertase inhibition in tumour cells.

First directed mutagenesis studies were performed on aIIb and a4 subunits. Mutating the basic residues of convertase recognition site prevented a subunit cleavage, but uncleaved aIIb^3 and a4^1 integrins were still able to mediate cell adhesion to their respective ligands [44, 45]. Despite of these disappointing results, some studies have been conducted later with other integrins. To examine the importance of cleavage, Delwel and collaborators introduced mutations in the cDNA encoding the RKKR sequence of the a6 subunit. In the human leukemia cells K562 used in this study, the a6A^1 integrin is expressed in a resting inactive conformation and need activation to bind ligand. The absence of cleavage of the a6A^1 integrin did not affected cell adhesion to laminin-1 after activation by the anti-^1 stimulatory antibody TS2/16. However, interestingly, when activation was performed with the phorbol ester PMA, only wild-type a6A^1 integrin was able to recognise laminin-1. The authors conclude that uncleaved a6A^1 is able of ligand binding and can transduce outside-in signals but fails in inside-out signalling as it can not be activated by phorbol ester [46, 47]. These results thus suggest that cleavage may provide for the flexibility required to allow proper conformational changes enabling the affinity modulation of the a6A^1 integrin.

The inhibition of PCs by specific inhibitors has also been used as an alternative strategy to elucidate the cleavage role in integrin function. In our laboratory we transfected the human colon adenocarcinoma cell line HT29-D4 with a vector encoding for the convertase inhibitor a1-PDX [48]. Clones of stable transfectants were further selected on the basis of their resistance to Pseudomonas Exotoxin A, a toxin activated upon cleavage by convertases. The expression of high levels of a 1-PDX inhibitor totally blocked the endoproteolytic processing of all the cleavable integrins subunits (a3, a6 and av) expressed in these cells. This leads to alterations in integrin function such as cell adhesion [48], cell migration [49], proliferation [50] and metastasis formation [51].

The absence of integrin processing has important consequences on signal trans-duction pathways initiated by ligation of av^5 integrin, leading to a reduced attachment to vitronectin. The reduced cell adhesion is most likely not due to changes in integrin affinity, but certainly reflects the inability of the uncleaved integrin to cluster or to interact with its partners [48]. The difference with Delwel results concerning this point [46, 47] could be due, either to a behaviour proper to each integrin, or to the already activated state of integrins in HT29-D4 cells.

Cells expressing uncleaved av integrins (PDX39P cells) also display a very motile behaviour on vitronectin when compared to control cells (PDX0 cells) and become able to invade collagen gels [49, 51]. The reduced adhesion due to the absence of proteolytic processing of av subunit could thus, at least partly, explain the increased motility of PDX39P cells. This is likely facilitated by alterations in cytoskeleton remodelling involving the av^5 integrin, the sole receptor for vitronectin in HT29-D4 [49].

Subcutaneous inoculation of cells in nude mice revealed that animals injected with a1-PDX-expressing cells exhibited delayed and lower incidence of tumour development, as well as reduced tumour size [50]. Expression of the convertase inhibitor also slightly decreased the tumorigenicity in immunosuppressed newborn rats (Figure 3) [51]. The reduced size of subcutaneous tumours in response to a1-PDX expression is likely due to differences in cell proliferation [50, 52].

However, in spite of their lower growth rate and the smaller tumours they produced, PDX39P cells exhibit a very aggressive behaviour when injected to immunosuppressed rats [51]. Indeed, the tumours they produce showed morphological evidence of higher local invasiveness and infiltrative pattern and produced about 10 times more metastases than control tumours (Figure 3).

The motile and invasive behaviour likely involves integrin a v^5 because a function-blocking mAb against the a v subunit was efficient to prevent both in vivo invasion and in vitro cell motility [51]. Taken together, all these results show that the cleavage of av subunit is essential for the function of av^5 integrin and has a marked impact on integrin-dependent events, especially those leading to cell migration. The molecular mechanism by which the endoproteolytic cleavage of a v subunit affects cell migration and aggressiveness is not precisely understood. Potential hypotheses include alterations in the cellular proteins associated with or regulated by av integrins. Thus, the unconventional processing of av by membrane type 1 matrix metalloproteinase (MT1-MMP), and the subsequent generation of the modified av^3 integrin, results in enhanced functional activity of the integrin [53, 54]. Moreover, further studies indicate that cells displaying the unconventional av^3 integrin and cells treated with the furin inhibitor dec-RVKR-cmk show an increased attachment to type I collagen [55]. In addition, a 1-PDX-expressing cells acquire the ability to invade collagen, a ligand for a2^1 integrin [51]. These results are consistent with furin-processed av controlling the cross-talk between av^3 and a2^1 integrins. Along with our own results, this clearly demonstrate that the cleavage of av subunit and the way it is cleaved can drastically influence the integrin function and consequently the behaviour of malignant cells.

The same strategy was used by others authors to demonstrate more broadly the role of PCs in tumour progression (for more details, see [56-58]). Using HT29 cells, Khatib and collaborators clearly demonstrated the role of convertases on activation of several elements involved in growth and malignant phenotype of tumours, such as the IGF-I receptor, plasminogen activators uPA and tPA and the receptor UPAR, or MT1-MMP [50, 56]. They observed that a1-PDX inhibits the IGF-I receptor processing, resulting in a default in IRS-1 signalling, an inhibition

Figure 3. PCs and tumor progression. (A) Macroscopic appearance of PDX39P- (a, b) and PDX0-induced tumours (c, d) three weeks after subcutaneous inoculation of cells in immunosuppressed newborn rats (B) Tumours volume was evaluated according to Stragand method Data are mean values ±SD from two experiments (total of 12 rats per cell line). suite. 1-PDX expression increases metastases formation. (C) Macroscopic appearance of the lungs from PDX39P- (a, b) and PDX0-inoculated rats (c, d) three weeks after injection of tumour cells. (D) Quantification of the number of lung metastases in PDX39P-and control cells-inoculated animals was done on each lung by three experimenters. Data represent mean values ±SD from two experiments (total of 12 rats per cell line). (E) Masson trichrome staining of paraffin embedded section. Micrograph (magnification, x200) shows voluminous metastases (M) with little healthy tissue. Pneumocytes (arrows) are pushed back beneath the pressure of metastatic cells. Reproduced from ref [51], with the authorisation of the American Journal of Pathology

Figure 3. PCs and tumor progression. (A) Macroscopic appearance of PDX39P- (a, b) and PDX0-induced tumours (c, d) three weeks after subcutaneous inoculation of cells in immunosuppressed newborn rats (B) Tumours volume was evaluated according to Stragand method Data are mean values ±SD from two experiments (total of 12 rats per cell line). suite. 1-PDX expression increases metastases formation. (C) Macroscopic appearance of the lungs from PDX39P- (a, b) and PDX0-inoculated rats (c, d) three weeks after injection of tumour cells. (D) Quantification of the number of lung metastases in PDX39P-and control cells-inoculated animals was done on each lung by three experimenters. Data represent mean values ±SD from two experiments (total of 12 rats per cell line). (E) Masson trichrome staining of paraffin embedded section. Micrograph (magnification, x200) shows voluminous metastases (M) with little healthy tissue. Pneumocytes (arrows) are pushed back beneath the pressure of metastatic cells. Reproduced from ref [51], with the authorisation of the American Journal of Pathology of IGF-1-dependent cell growth and consequently a reduced tumour size [50]. Tumour growth was also reduced in Scid mice inoculated with a1-PDX-transfected astrocytoma cells certainly due to a decrease in tumour cell proliferation [59]. Expression of the inhibitor in neck squamous cell carcinoma cells also reduced in vitro cell invasion and in vivo invasion across tracheal wall. After injection of these cells in Scid mice, tumours appeared late and remained smaller than with control cells [60].

However, all these results did not take the integrin cleavage status into consideration. Generally, the use of PCs inhibitors is not appropriate to elucidate the role of integrin cleavage on tumour invasion because of the large targets of conver-tases also involved in this process: metalloproteinases as MT-MMPs, growth factor as TGFP, growth factor receptors as IGF1-receptor... The av integrin plays an essential role in tumourigenicity of M21 melanoma cells [61]. We thus transfected both wild-type and mutant av cDNAs into av-negative M21-L melanoma cells provided by Dr. David A. Cheresh (Scripps Clinic, La Jolla, USA). Using collagen gel invasion assays, we confirmed that the blockage of av subunit processing led to an invasive phenotype, as described above for a1-PDX-expressing cells (unpublished results). Overall, our findings indicate that the endoproteolytic processing of av subunit obviously affects the function of avP5 integrin and actively contribute to the malignant phenotype of human tumour cells. The molecular mechanism of the increased motility supported by av-containing integrins and the aggressiveness that results from it remains to be determined.

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